A short story of 3AB-OS cancer stem cells, a possible model for studying cancer stemness

Cancer Stem Cells (CSCs) are thought to be the cause of cancer initiation, growth and development. Thus, a challenge in cancer research is their identification and eradication. In our laboratory, by chemical treatment of the human osteosarcoma (OS) MG63 cell line, we have isolated and characterized 3AB-OS cells, a human OS CSC line. 3AB-OS cells transdifferentiate in vitro into cells of the three derivatives germ layers and, when xenografted in athymic mice they are highly tumorigenic and recapitulate in vivo crucial features of human OS. They even express a reprogrammed energy metabolism, with a dependence on glycolytic metabolism more strong than parental MG63 cells. 3AB-OS cells have chromosomes showing a great number of abnormalities which are very similar to abnormalities found in both pediatric and adult osteosarcomas. In comparison with parental MG63 cells (where TP53 gene is hypermethylated, rearranged and in single copy), 3AB-OS cells have TP53 gene unmethylated, rearranged and in multiple copies. Moreover, the mutp53 (p53-R248W/P72R) is post-translationally stabilized, has nuclear localization and a gain of function. A great number of results obtained in our laboratories suggested that p53 mutation could be the “driver mutation” at the origin of the transformation of MG63 cells into 3AB-OS CSCs

Parthenolide induces caspase-independent and AIF-mediated cell death in human osteosarcoma and melanoma cells.

The mechanism of the cytotoxic effect exerted by parthenolide on tumor cells is not clearly defined today. This article shows that parthenolide stimulates in human osteosarcoma MG63 and melanoma SK-MEL-28 cells a mechanism of cell death, which is not prevented by z-VAD-fmk and other caspase inhibitors. In particular treatment with parthenolide rapidly stimulated (1-2 h) reactive oxygen species (ROS) generation by inducing activation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and NADPH oxidase. This event caused depletion of thiol groups and glutathione, NF-κB inhibition, c-Jun N-terminal kinase (JNK) activation, cell detachment from the matrix, and cellular shrinkage. The increase of ROS generation together with the mitochondrial accumulation of Ca(2+) also favored dissipation of Δψm, which seemed primarily determined by permeability transition pore opening, since Δψm loss was partially prevented by the inhibitor cyclosporin A. Staining with Hoechst 33342 revealed in most cells, at 3-5 h of treatment, chromatin condensation, and fragmentation, while only few cells were propidium iodide (PI)-positive. In addition, at this stage apoptosis inducing factor (AIF) translocated to the nucleus and co-localized with areas of condensed chromatin. Prolonging the treatment (5-15 h) ATP content declined while PI-positive cells strongly augmented, denouncing the increase of necrotic effects. All these effects were prevented by N-acetylcysteine, while caspase inhibitors were ineffective. We suggest that AIF exerts a crucial role in parthenolide action. In accordance, down-regulation of AIF markedly inhibited parthenolide effect on the production of cells with apoptotic or necrotic signs. Taken together our results demonstrate that parthenolide causes in the two cell lines a caspase-independent cell death, which is mediated by AIF

Axolotl Ambystoma mexicanum extract induces cell cycle arrest and differentiation in human acute myeloid leukemia HL-60 cells

Acute myeloid leukemia is the most common form of acute leukemia in adults, constituting about 80% of cases. Although remarkable progress has been made in the therapeutic scenario for patients with acute myeloid leukemia, research and development of new and effective anticancer agents to improve patient outcome and minimize toxicity is needed. In this study, the antitumor activity of axolotl (AXO) Ambystoma mexicanum crude extract was assessed in vitro on the human acute myeloid leukemia HL-60 cell line. The anticancer activity was evaluated in terms of ability to influence proliferative activity, cell viability, cell cycle arrest, and differentiation. Moreover, gene expression analysis was performed to evaluate the genes involved in the regulation of these processes. The AXO crude extract exhibited antiproliferative but not cytotoxic activities on HL-60 cells, with cell cycle arrest in the G0/G1 phase. Furthermore, the AXO-treated HL-60 cells showed an increase in both the percentage of nitroblue tetrazolium positive cells and the expression of CD11b, whereas the proportion of CD14-positive cells did not change, suggesting that extract is able to induce differentiation toward the granulocytic lineage. Finally, the treatment with AXO extract caused upregulation of CEBPA, CEBPB, CEBPE, SPI1, CDKN1A, and CDKN2C, and downregulation of c-MYC. Our data clearly show the potential anticancer activity of Ambystoma mexicanum on HL-60 cells and suggest that it could help develop promising therapeutic agents for the treatment of acute myeloid leukemia.peer-reviewe

Anticancer effects of an extract from a local planarian species on human acute myeloid leukemia HL-60 cells in vitro

Current anti-cancer drugs can cause many undesirable side effects to patients. Thus, there is a constant need to develop alternative therapeutic drugs. Bioactive compounds derived from natural products including animals, plants and microorganisms are being actively studied as sources for anticancer treatments. Freshwater planarians are important models for stem cell research and regeneration. However, to date, no studies on the biological activities of planaria extracts on cancer have been published. The aim of this study was to examine the potential antitumoral activity of the extract from planaria species-Malta (PSM) on human acute myeloid leukemia (AML) HL-60 cells. Antiproliferative activity was studied in terms of proliferation, apoptosis and differentiation. The expression of genes involved in the regulation of these important cellular processes was also analyzed using real-time PCR. PSM extract exhibited a selective cytotoxic effect on HL-60 cells when compared to normal lymphocytes. Furthermore, cell cycle analysis and Annexin V/PI assay showed that the extract induced apoptosis in HL-60 cells. The PSM extract induced myeloid differentiation with HL-60 cells showing a decreased nucleo/cytoplasmic ratio, an increase in nitroblue tetrazolium-positive cells, and CD11b- and CD14-positive cells. Finally, we also found that the PSM extract increased the expression of CEBPA, CEBPB, CEBPE, SPI1, BAX, CDKN1A and CDKN2C; whereas it reduced the expression of c-MYC and BCL2. This is the first study to reveal the antiproliferative, cytotoxic, and differentiation potential of PSM on HL-60 cells and suggests that it may have considerable potential for development as a novel natural product-based anticancer agent against AML.peer-reviewe

Parthenolide induces superoxide anion production by stimulating EGF receptor in MDA-MB-231 breast cancer cells.

The sesquiterpene lactone parthenolide (PN) has recently attracted considerable attention because of its anti-microbial, anti-inflammatory and anticancer effects. However, the mechanism of its cytotoxic action on tumor cells remains scarcely defined. We recently provided evidence that the effect exerted by PN in MDA-MB-231 breast cancer cells was mediated by the production of reactive oxygen species (ROS). The present study shows that PN promoted the phosphorylation of EGF receptor (phospho-EGFR) at Tyr1173, an event which was observed already at 1  h of incubation with 25  µM PN and reached a peak at 8-16  h. This effect seemed to be a consequence of ROS production, because N-acetylcysteine (NAC), a powerful ROS scavenger, prevented the increment of phospho-EGFR levels. In addition fluorescence analyses performed using dihydroethidium demonstrated that PN stimulated the production of superoxide anion already at 2-3  h of incubation and the effect further increased prolonging the time of treatment, reaching a peak at 8-16  h. Superoxide anion production was markedly hampered by apocynin, a well known NADPH oxidase (NOX) inhibitor, suggesting that the effect was dependent on NOX activity. The finding that AG1478, an EGFR kinase inhibitor, substantially blocked both EGFR phosphorylation and superoxide anion production strongly suggested that phosphorylation of EGFR can be responsible for the activation of NOX with the consequent production of superoxide anion. Therefore, EGFR phosphorylation can exert a key role in the production of superoxide anion and ROS induced by PN in MDA-MB-231 cells

Involvement of PAR-4 in Cannabinoid-Dependent Sensitization of Osteosarcoma Cells to TRAIL-Induced Apoptosis

The synthetic cannabinoid WIN 55,212-2 is a potent cannabinoid receptor agonist with anticancer potential. Experiments were performed to determine the effects of WIN on proliferation, cell cycle distribution, and programmed cell death in human osteosarcoma MG63 and Saos-2 cells. Results show that WIN induced G2/M cell cycle arrest, which was associated with the induction of the main markers of ER stress (GRP78, CHOP and TRB3). In treated cells we also observed the conversion of the cytosolic form of the autophagosome marker LC3-I into LC3-II (the lipidated form located on the autophagosome membrane) and the enhanced incorporation of monodansylcadaverine and acridine orange, two markers of the autophagic compartments such as autolysosomes. WIN also induced morphological effects in MG63 cells consisting in an increase in cell size and a marked cytoplasmic vacuolization. However, WIN effects were not associated with a canonical apoptotic pathway, as demonstrated by the absence of specific features, and only the addition of TRAIL to WIN-treated cells led to apoptotic death probably mediated by up-regulation of the tumor suppressor factor PAR-4, whose levels increased after WIN treatment, and by the translocation of GRP78 on cell surface

(In)Distinctive Role of Long Non-Coding RNAs in Common and Rare Ovarian Cancers

Simple Summary:& nbsp;Ovarian cancers (OCs) are the most lethal form of gynecological tumors. The commonest are high-grade serous OCs, while rare OCs originate from many different cell types, such as epithelial, germ cell, sex cord-stromal, or mixed types. Rare OCs have distinct molecular characteristics, prognosis, and therapeutic approaches. However, all ovarian malignancies mostly share the same problem: late diagnosis due to the lack of specific symptoms. Therefore, there is a perpetual need to discover better diagnostic, prognostic, and predictive biomarkers, as well as new therapeutic approaches. In recent years, long non-coding RNAs (lncRNAs) have gained widespread attention because of their important role in various biological pathways. They have multiple mechanisms of action with an important role in many cellular processes related to OCs development and progression. This review will focus on the different aspects of lncRNAs in OCs and attempt to highlight the distinctive role of lncRNAs in common and rare OCs

Gynocare cost action ca18117 “European network for gynaecological rare cancer research : from concept to cure”

Introduction: Up to 50% of all gynecologic tumors can be classified as rare (incidence of < 6 per 100,000 women) and usually have a poor prognosis owing to delayed diagnosis and treatment. Contrary to other common solid tumors, the treatment of rare gynecologic tumors (RGT) is often based on retrospective studies, expert opinion or extrapolation from other tumor sites with similar histology, leading to difficulty in developing guidelines for clinical practice. Currently, gynecologic cancer research, due to distinct scientific and technological challenges, is lagging behind. Moreover, the overall efforts for addressing these challenges are fragmented across different European countries and indeed, worldwide. Aims: GYNOCARE is an EU funded programme that aims to address these challenges by creating a unique network between key stakeholders covering distinct RGT research areas ranging from concept to cure: basic research, biobanking, bridging with industry, and setting up the legal and regulatory requirements for international innovative clinical trials. Methods: To achieve these ambitious goals, GYNOCARE focuses on (1) capacity-building on rare gynaecological cancer by connecting high-quality scientific communities in various disciplines, existing networks, policy-makers, industrial partners, and patient organisations across Europe and beyond; (2) coordinating, and contributing to the development of a research roadmap dedicated to connect (innovative) basic research to (harmonised) biobanking to ‘smarter’ clinical trials; (3) the development of a platform for sharing best practices, including funding roadmap and legal/ethical requirements, in gynaecological cancers – aiming to advice policy-makers and other key stakeholders; and (4) providing (equal) networking opportunities for early-stage researchers, and other talented young professionals. Results: Over 50 members from 20 countries form part of the GYNOCARE Consortium. Conclusion: This COST Action is an effective tool in order to bring together different key stakeholders in the field of rare gynecologic cancer.peer-reviewe

Parthenolide generates reactive oxygen species and autophagy in MDA-MB231 cells. A soluble parthenolide analogue inhibits tumour growth and metastasis in a xenograft model of breast cancer.

Triple-negative breast cancers (TNBCs) are clinically aggressive forms associated with a poor prognosis. We evaluated the cytotoxic effect exerted on triple-negative MDA-MB231 breast cancer cells both by parthenolide and its soluble analogue dimethylamino parthenolide (DMAPT) and explored the underlying molecular mechanism. The drugs induced a dose- and timedependent decrement in cell viability, which was not prevented by the caspase inhibitor z-VAD-fmk. In particular in the first hours of treatment (1–3 h), parthenolide and DMAPT strongly stimulated reactive oxygen species (ROS) generation. The drugs induced production of superoxide anion by activating NADPH oxidase. ROS generation caused depletion of thiol groups and glutathione, activation of c-Jun N-terminal kinase (JNK) and downregulation of nuclear factor kB (NF-kB). During this first phase, parthenolide and DMAPT also stimulated autophagic process, as suggested by the enhanced expression of beclin-1, the conversion of microtubule-associated protein light chain 3-I (LC3-I) to LC3-II and the increase in the number of cells positive to monodansylcadaverine. Finally, the drugs increased RIP-1 expression. This effect was accompanied by a decrement of pro-caspase 8, while its cleaved form was not detected and the expression of c-FLIPS markedly increased. Prolonging the treatment (5–20 h) ROS generation favoured dissipation of mitochondrial membrane potential and the appearance of necrotic events, as suggested by the increased number of cells positive to propidium iodide staining. The administration of DMAPT in nude mice bearing xenografts of MDA-MB231 cells resulted in a significant inhibition of tumour growth, an increment of animal survival and a marked reduction of the lung area invaded by metastasis. Immunohistochemistry data revealed that treatment with DMAPT reduced the levels of NF-kB, metalloproteinase-2 and -9 and vascular endothelial growth factor, while induced upregulation of phosphorylated JNK. Taken together, our data suggest a possible use of parthenolide for the treatment of TNBCs

The oxygen radicals involved in the toxicity induced by parthenolide in MDA-MB-231 cells

It has been shown that the sesquiterpene lactone parthenolide lowers the viability of MDA-MB-231 breast cancer cells, in correlation with oxidative stress. The present report examined the different radical species produced during parthenolide treatment and their possible role in the toxicity caused by the drug. Time course experiments showed that in the first phase of treatment (0-8 h), and in particular in the first 3 h, parthenolide induced dichlorofluorescein (DCF) signal in a large percentage of cells, while dihydroethidium (DHE) signal was not stimulated. Since the effect on DCF signal was suppressed by apocynin and diphenyleneiodonium (DPI), two inhibitors of NADPH oxidase (NOX), we suggest that parthenolide rapidly stimulated NOX activity with production of superoxide anion (O2•-), which was converted by superoxide dismutase 1 (SOD1) into hydrogen peroxide (H2O2). In the second phase of treatment (8-16 h), parthenolide increased the number of positive cells to DHE signal. Since this event was not prevented by apocynin and DPI and was associated with positivity of cells to MitoSox Red, a fluorochrome used to detect mitochondrial production of O2•-, we suggest that parthenolide induced production of O2•- at the mitochondrial level independently by NOX activity in the second phase of treatment. Finally, in this phase, most cells became positive to hydroxyphenyl fluorescein (HPF) signal, a fluorescent probe to detect highly reactive oxygen species (hROS), such as hydroxyl radical and peroxynitrite. Therefore, parthenolide between 8-16 h of treatment induced generation of O2•- and hROS, in close correlation with a marked reduction in cell viability